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Abstract

The insular cortex (IC) is an important forebrain structure involved in pain perception
and taste memory formation. Using a 64-channel multi-electrode array system, we recently
identified and characterized two major forms of synaptic plasticity in the adult mouse
IC: long-term potentiation (LTP) and long-term depression (LTD). In this study, we
investigate injury-related metaplastic changes in insular synaptic plasticity after
distal tail amputation. We found that tail amputation in adult mice produced a selective
loss of low frequency stimulation-induced LTD in the IC, without affecting (RS)-3,5-dihydroxyphenylglycine
(DHPG)-evoked LTD. The impaired insular LTD could be pharmacologically rescued by
priming the IC slices with a lower dose of DHPG application, a form of metaplasticity
which involves activation of protein kinase C but not protein kinase A or calcium/calmodulin-dependent
protein kinase II. These findings provide important insights into the synaptic mechanisms
of cortical changes after peripheral amputation and suggest that restoration of insular
LTD may represent a novel therapeutic strategy against the synaptic dysfunctions underlying
the pathophysiology of phantom pain.

Background

Insular cortex (IC) is an integrating forebrain structure involved in several sensory
and cognitive functions, such as interoceptive awareness, taste memory, and pain perception
[1-3]. In particular, human brain imaging studies have demonstrated the activation of IC
in a broad range of pain conditions [4-6]. Moreover, electrical stimulation of IC directly elicits painful sensations in human
subjects [7-9]. The involvement of IC in chronic pain has also been confirmed by animal experiments,
showing the presence of nociceptive neurons [10,11] and pain-evoked biochemical changes [12,13] in this area. Genetic [14,15] or pharmacological [16-19] manipulation of the IC could alter the pain sensitivity. Importantly, long-term potentiation
(LTP) has been revealed in the IC by both in vivo[20,21] and in vitro[22,23] electrophysiological recordings. Furthermore, neuropathic pain experience could occlude
the electrical induction of insular LTP in adult mice [18], suggesting that chronic pain may share common mechanisms with insular synaptic plasticity
[24].

Phantom pain refers to the feeling of pain in a body part that has been amputated
[25-27]. Mechanistically, limb amputation has been shown to cause dramatic cortical reorganization
in humans and primates [28-31], the amount of which correlates well with the extent of phantom pain in some reports
[32-34]. We previously demonstrated that digit amputation in rats or tail amputation in mice
triggered long-lasting plastic alterations in the anterior cingulate cortex (ACC),
including an enhancement of excitatory synaptic responses in vivo[35,36], loss of long-term depression (LTD) in vitro[37,38] and activation of activity-dependent immediate early genes [37,39]. In addition to ACC, human imaging studies also revealed a correlation between the
IC activation and phantom pain [25,40,41]. Thus, it is important to investigate the possible changes in synaptic plasticity
in the IC after amputation.

It is believed that peripheral injury elicits long-lasting plastic changes in the
brain via at least two major mechanisms: one is direct enhancement of excitatory synaptic
transmission, and the other is loss of the ability to undergo LTD [42], also see Table 1]. In the present study, we used a 64-channel multi-electrode dish (MED64) recording
system [23,38,43] to examine injury-related metaplastic changes in insular LTD caused by tail amputation
in the adult mice. Our previous results demonstrated the co-existence of two different
forms of LTD in the IC: NMDA receptor-dependent LTD and NMDA receptor-independent
LTD [44]. Here, we report that tail amputation produces a selective loss of low frequency
stimulation (LFS)-induced LTD in the adult mice IC, leaving (RS)-3,5-dihydroxyphenylglycine
(DHPG)-evoked LTD intact. The impaired insular LTD could be pharmacologically rescued
by priming the IC slices with application of a low-dose group I metabotropic glutamate
receptor (mGluR) agonist DHPG, a form of metaplasticity that involves activation of
protein kinase C (PKC) but not protein kinase A (PKA) or calcium/calmodulin-dependent
protein kinase II (CaMKII).

Table 1.Summary of previous studies on injury-evoked changes in the induction of LTD

Results

Loss of LFS-evoked LTD in the IC after tail amputation

Our previous work has demonstrated that digit or tail amputation in rats or mice could
abolish the induction of LFS-evoked LTD in the ACC [37,38]. Here, we employed a previously-established 64-channel multi-electrode array system,
i.e. the MED64 system [23,38,44], to examine whether the insular synaptic plasticity is equally sensitive to the amputation-induced
peripheral injury in adult mice. MED64 recordings were performed in the IC slices
obtained from sham control or tail-amputated mice at 2 weeks after surgery (Figure 1A). The relative location of the MED64 probe within the IC slice is shown in Figure 1B. We focused our recording sites on the rostral IC region at the level of the corpus
callosum connection, where the stimulation site is usually located in the deep layer
(layer V-VI) of the IC slice (the red dot in Figure 1B). One representative example recording is illustrated in Figure 1C (before LFS) and Figure 1D (60 min after LFS) for the tail-amputated group. It is clearly discerned that LFS
failed to induce any depression of field excitatory postsynaptic potential (fEPSP)
in this slice. The averaged data showed a complete loss of LFS-evoked LTD in the superficial
layer (96.8 ± 1.9% of baseline at 60 min after LFS, n = 9 slices/5 mice, P = 0.701, Student’s t-test, Figure 1E). In contrast, the sham control group exhibited clear LTD of multisite synaptic
responses (66.2 ± 2.5% of baseline, n = 9 slices/7 mice, P < 0.001, Student’s t-test, Figure 1E), which is consistent with our previous publication [44].

Figure 1.Loss of LFS-evoked LTD in the superficial layer of the IC after tail amputation. (A) A schematic diagram showing the tail amputation model (up) and the experimental procedure
(lower). All insular slices are obtained at 2 weeks after amputation in the present
study. (B) Light microscopy photograph showing the relative location of the IC slice with the
MED64 probe, the stimulation site (red dot) and the layer designation. (C and D) An overview of the 64-channel multi-electrode array recordings in the tail-amputated
IC slice (C: before LFS; D: 60 min after LFS). No synaptic depression was revealed. Red dots mark the stimulation
sites in the deep layer. Calibration: 100 μV, 10 ms. (E) Pooled data of LFS-elicited LTD in the superficial layer of the IC for sham control
(n = 9 slices/7 mice) and tail amputation (n = 9 slices/5 mice) groups. The sham group
showed typical LTD lasting for 1 h, while tail amputation abolished the LTD induction.
Sample fEPSP recordings taken at the times indicated by the corresponding numbers
are shown above the plot. Calibration: 100 μV, 10 ms. Horizontal bars denote the period
of LFS delivery. Error bars represent SEM.

Neurons in different layers of the IC are considered to have different afferent and
efferent connections with other areas of the brain, and thus may mediate distinct
functions [48-50]. Therefore, we next asked whether tail amputation could also affect the LTD induction
in the deep layer as it did in the superficial layer. As previously described [44], LFS application produced a long-lasting synaptic depression of fEPSPs recorded in
the deep layer of the IC from the sham control group (70.3 ± 1.5% of baseline at 60 min
after LFS, n = 9 slices/7mice, P < 0.001, Student’s t-test, Figure 2A). However, LFS failed to induce any LTD in the tail-amputated group (94.0 ± 2.6%
of baseline, n = 9 slices/5 mice, P = 0.137, Student’s t-test, Figure 2A). Statistical analysis revealed a strong significant difference between sham and
tail-amputated groups in the degree of LTD in both superficial layer (P < 0.001, Student’s t-test, Figure 1E) and deep layer (P < 0.001, Student’s t-test, Figure 2A).

Figure 2.Loss of LFS-evoked LTD in the deep layer of the IC after tail amputation. (A) Pooled data of LFS-elicited LTD in the deep layer of the IC for sham control (n = 9
slices/7 mice) and tail amputation (n = 9 slices/5 mice) groups. Tail amputation also
abolished the LTD induction in the deep layer. Sample fEPSP recordings taken at the
times indicated by the corresponding numbers are shown above the plot. Calibration:
100 μV, 10 ms. Horizontal bars denote the period of LFS delivery. (B) Bar histogram showing the induction ratio of LTD (the percentage of LTD-showing channels
among all activated channels) in the superficial layer and deep layer of the IC for
sham control (n = 9 slices/7 mice) and tail-amputated (n = 9 slices/5 mice) groups.
***P < 0.001. Error bars represent SEM.

Lack of the effect of amputation on DHPG-induced insular LTD

Recently, we reported the co-existence of two distinct forms of LTD in the insular
synapses: NMDA receptor-dependent LTD induced by LFS, and NMDA receptor-independent
LTD induced by DHPG application [44]. Next, we sought to examine whether tail amputation could also affect the induction
of DHPG-LTD. We induced DHPG-LTD by bath application of 100 μM DHPG for 20 min and
then washed it out to monitor the course of chemically-induced LTD for 50 min. Similar
to the previous study, DHPG infusion produced a rapid and long-lasting depression
of fEPSP in the IC slices (Figure 3A and B). The synaptic responses of the superficial layer were reduced to 72.5 ± 1.8%
of baseline (n = 7 slices/7 mice, P < 0.001, Student’s t-test, Figure 3A) at 50 min after washout of DHPG in the sham group. Interestingly, we did not observe
any abolition of DHPG-LTD in the IC after tail amputation (73.6 ± 2.1% of baseline,
n = 9 slices/9 mice, P < 0.001, Student’s t-test, Figure 3A). Similarly, the lack of effect of amputation on DHPG-LTD is also replicated in
the deep layer of the IC (sham control: 77.1 ± 3.0% of baseline, n = 8 slices/8 mice,
P < 0.001, Student’s t-test; tail amputation: 73.3 ± 2.0% of baseline, n = 9 slices/9
mice, P < 0.001, Student’s t-test, Figure 3B). The magnitude and duration of DHPG-LTD in the tail-amputated group did not differ
from the sham control (superficial layer: P = 0.311, Student’s t-test, Figure 3A; deep layer, P = 0.303, Student’s t-test, Figure 3B). Likewise, the induction ratio of DHPG-LTD in the IC was not different between
the two groups in either superficial layer (sham vs. tail amputation: 49.2 ± 10.1%
vs. 47.6 ± 6.7%, P = 0.431, Student’s t-test) or deep layer (sham vs. tail amputation: 63.6 ± 8.1% vs.
59.6 ± 6.9%, P = 0.711, Student’s t-test, Figure 3C). Taken together, these results suggest that tail amputation selectively blocked
the induction of LFS-evoked insular LTD, with the DHPG-LTD being intact. This result
in the IC is in contrast to that in the ACC, where tail amputation prevented the occurrence
of both LFS-induced LTD and mGluR1-mediated LTD [38].

Enhanced synaptic transmission in the IC after tail amputation

It has been previously reported that peripheral inflammation or nerve injury could
trigger a long-term enhancement of excitatory synaptic transmission in various brain
regions, such as ACC [51-54], amygdala [55-57], and hippocampus [58]. We next examined whether similar alterations in synaptic efficacy could be elicited
in the IC after peripheral injury. The input–output relationships, measuring fEPSP
slope (output) as a function of the afferent stimulus intensity (input), were compared
between sham control and tail-amputated (two weeks) groups. The slope of the curve
was evidently shifted to the left at higher stimulation intensities after amputation
(n = 6 slices/4 mice for both superficial layer and deep layer), compared with that
in control group (n = 6 slices/6 mice for superficial layer; n = 5 slices/5 mice for
deep layer) (Figure 4A and B). These results suggest that excitatory synaptic transmission is likely enhanced
following tail amputation experience. Nevertheless, the curves did not move leftward
in a parallel manner, indicating no alteration in the threshold for inducing fEPSPs.
Furthermore, the input–output curves of the total number of activated channels showed
a similar trend between the two groups (n = 6 slices/6 mice for sham control; n = 8
slices/5 mice for tail-amputated group; Figure 4C).

Figure 4.Enhancement of synaptic transmission in the IC after tail amputation. (A) The input–output relationship of the fEPSP slope in the superficial layer of the
IC. Shown are the percentage changes of the fEPSP slope (normalized to the slope value
at 8 μA) in response to series of ascending stimulation intensities. Tail amputation
(n = 6 slices/4 mice) caused a leftward shift of the input–output curve compared to
the sham control group (n = 6 slices/6 mice). (B) Pooled data of the input–output relationship of the fEPSP slope in the deep layer
of the IC. Similarly, tail amputation (n = 6 slices/4 mice) resulted in a leftward
shift of the curve compared to the sham control (n = 5 slices/5 mice). The insets
in (A) and (B) show the representative fEPSP traces recorded at 18 μA for both sham
(left) and tail-amputated (right) groups. Calibration: 100 μV, 10 ms. (C) The input-out curve of the number of activated channels obtained at graded stimulation
intensities in the IC slice. Significant difference was detected between the sham
control (n = 6 slices/6 mice) and tail-amputated (n = 8 slices/5 mice) group at higher
stimulation intensities. Error bars represent SEM.

Prior activation of group I mGluRs could produce metaplastic effects on synaptic plasticity
in the hippocampus, shown as a large enhancement in the induction of hippocampal LTP
[59,60], for review, see [61]. Our previous work revealed another form of group I mGluR-mediated metaplasticity
in the ACC, that is, priming ACC slices with pharmacological activation of mGluR1
rescued the loss of LTD caused by the tail amputation [38]. Here, using the same rationale, we attempted to rescue LFS-induced insular LTD by
priming the IC slices with bath application of a lower dose of DHPG (20 μM, 20 min).
Figure 5A and B illustrates the overview of the 64-channel recordings obtained before LFS
and 60 min after LFS in one DHPG-primed and tail-amputated IC slice. DHPG treatment
at this dose failed to trigger any LTD of multisite synaptic responses, but only a
rapid and transient acute depression was observed in either superficial layer (90.8%
of baseline at the end of DHPG infusion, Figure 5C) or deep layer (92.4% of baseline at the end of DHPG infusion, Figure 5D). However, subsequent LFS indeed led to a significant depression of the fEPSPs in
a single example (superficial layer: 70.0% of baseline at 60 min after LFS, Figure 5C; deep layer: 65.9% of baseline at 60 min after LFS, Figure 5D) and in pooled data (superficial layer: 67.5 ± 3.5% of baseline, n = 6 slices/5
mice, P = 0.002, Student’s t-test, Figure 5E; deep layer: 67.5 ± 2.9% of baseline, n = 5 slices/5 mice, P = 0.008, Student’s t-test, Figure 5F). The magnitude of DHPG-rescued LTD in the tail-amputated mice is similar to that
of the sham control mice (compare Figure 5E and F with Figure 1E and Figure 2A). These results indicate that similar to the ACC synapses, prior activation of group
I mGluRs can produce a form of metaplasticity that restores the LFS-evoked LTD in
the IC in the tail-amputated mice.

Figure 5.Pharmacological rescue of LFS-evoked insular LTD in the tail-amputated mice by group
I mGluR activation. (A and B) An overview of 64-channel multi-electrode array recordings in the tail-amputated
IC slice (A: before LFS; B: 60 min after LFS). DHPG (20 μM) was applied for 20 min followed by washout for 30 min.
Then LFS was given. Bath application of low dose of DHPG only elicited acute depression.
However, subsequent LFS induced clear LTD after priming with DHPG. Red dots denote
the stimulation sites in the deep layer. Calibration: 100 μV, 10 ms. (C and D) One representative example slice showing the rescue of LFS-induced LTD by DHPG priming
in both superficial layer (C) and deep layer (D) of the IC. Inset traces show representative fEPSPs at the time points indicated by
the numbers in the graph. Calibration: 100 μV, 10 ms. (E and F) Summarized data for the superficial layer (n = 6 slices/5 mice) and deep layer (n = 5
slices/5 mice). Horizontal bars denote the period of DHPG application or LFS delivery
as indicated. Error bars represent SEM.

Protein kinase C, but not CaMKII or PKA, is involved in the rescue of insular LTD

To probe the mechanisms underlying the metaplastic rescue of LFS-evoked LTD in the
IC, we next performed pharmacological experiments using different protein kinase inhibitors,
based on previous reports showing the critical roles of various protein kinases in
mediating multiple forms of metaplasticity in the hippocampus [for reviews, see [61,62]. At first, we examined the involvement of PKC in the DHPG-induced priming effect,
given the increasing evidence supporting the role of PKC in metaplasticity [63-65]. Co-application of a PKC inhibitor chelerythrine (Che, 3 μM) with the DHPG (20 μM,
20 min) prevented the rescue of LTD in both superficial layer (96.2 ± 2.4% of baseline
within the last 10 min of recording, n = 6 slices/6 mice, P = 0.006, One-Way ANOVA followed by Fisher’s LSD test, Figure 6B and E) and deep layer (96.0 ± 1.4% of baseline within the last 10 min of recording,
n = 6 slices/6 mice, P < 0.001, One-Way ANOVA followed by Fisher’s LSD test, Figure 7B and E) of the IC slice taken from tail-amputated mice. In contrast, simultaneous
treatment of the IC slice with vehicle had no effect on the LTD rescue (superficial
layer: 71.0 ± 2.9% of baseline, n = 5 slices/3 mice, Figure 6A and E; deep layer: 79.3 ± 1.9% of baseline, n = 5 slices/3 mice, Figure 7A and E).

Besides PKC, CaMKII and PKA have also been shown to mediate certain forms of metaplasticity
[66-68]. Therefore, we also evaluated the role of these two kinases in DHPG-rescued insular
LTD in the tail-amputated mice. As shown in Figure 6C-E, neither KN62 (5 μM, a CaMKII inhibitor) nor KT5720 (1 μM, a PKA inhibitor) could
block the induction of LTD in the superficial layer of the IC (KN62: 65.0 ± 3.5% of
baseline, n = 6 slices/5 mice, P = 0.595, One-Way ANOVA followed by Fisher’s LSD test; KT5720: 72.6 ± 3.9% of baseline,
n = 7 slices/5 mice, P = 0.558, One-Way ANOVA followed by Fisher’s LSD test). Similar results were obtained
in the deep layer (KN62: 72.8 ± 2.6% of baseline, n = 6 slices/5 mice, P = 0.382, One-Way ANOVA followed by Fisher’s LSD test; KT5720: 81.9 ± 1.9% of baseline,
n = 7 slices/5 mice, P = 0.544, One-Way ANOVA followed by Fisher’s LSD test, Figure 7C-E). These observations are consistent with our previous results in the ACC [38], suggesting that PKC, but not CaMKII or PKA, acts as a major mediator in mGluR-evoked
metaplasticity in the IC in tail-amputated animals.

Discussion

There is considerable evidence indicating the critical role of the IC in pain perception
and memory storage [2,3,16-18]. However, few studies have been conducted at the cellular level to address the synaptic
basis of IC-mediated higher brain functions. Our recent work demonstrates that fast
excitatory synaptic transmission in the IC is mainly mediated by postsynaptic AMPA/kainate
receptors and that both LTP and LTD could be induced reliably but with different receptor
mechanisms [23,44,48]. Since cortical plasticity has been proposed to be an endpoint measurement and working
mechanism of chronic pain [24,69], it would be interesting to address the metaplastic effects of chronic pain experience
in vivo on the induction of insular LTP and LTD in vitro. We recently report that nerve injury-induced neuropathic pain could fully occlude
the subsequent induction of LTP in the IC [18]. In the present study, using a 64-channel multi-electrode array system, we further
evaluated the effect of abnormal pain processing on insular LTD. We found that a two-week
experience of amputation-induced peripheral injury resulted in a selective impairment
of insular LTD induction by the LFS protocol, but without any effect on DHPG-induced
LTD. Priming the IC slices with pharmacological activation of group I mGluRs rescued
the LFS-induced LTD after amputation, which involves the activation of PKC, but not
PKA or CaMKII.

Loss of LTD in the IC after amputation

One of the central findings in this study is the loss of LFS-evoked LTD in tail-amputated
IC slices. We selected two weeks after amputation as the time point for taking the
IC slices for multi-channel recordings, mainly based on our previous publications
showing the occurrence of marked plastic changes in the ACC at this time. Specifically,
we found that peripheral amputation abolished LTD and enhanced extracellular signal-regulated
kinase activation in the rodent ACC at two weeks [37-39]. Nevertheless, amputation-caused plastic changes in the brain might be time-dependent.
For example, digit amputation can abolish ACC LTD and enhance hippocampal LTP at 45 min
but failed to elicit any significant change in the hippocampus at 20 min or earlier
[37,45]. Thus, future studies are clearly needed to investigate if tail amputation-induced
loss of insular LTD is time dependent, and if so, when the metaplastic alterations
are initiated and how long they can last.

The detailed mechanisms underlying this LTD abolishment are not well understood. However,
our previous work revealed a similar deficit of LTD induction in the ACC from adult
rats or mice subjecting to digit or tail amputation, respectively [37,38]. In addition, tissue amputation produced a rapid and prolonged enhancement of sensory
responses to noxious stimulation, dramatic membrane depolarization, as well as large-scale
expression of several immediate early genes and signaling molecules in the ACC [35,37,39,70], for review, see [42]. These observations allow us to speculate that enhanced postsynaptic excitability
might also occur in the IC after tail amputation, which leads to the failure of LTD
induction. Supporting this assertion, we found a leftward shift of input–output curves
of fEPSPs in tail-amputated slices as compared to the control group. Furthermore,
our recent work demonstrates that induction of insular LTD by LFS involves activation
of the NMDA receptor and mGluR5 [44]. Since DHPG-induced LTD is not affected by amputation (see below), an alternative
explanation for the loss of LFS-evoked LTD might be due to the changes in the expression
and/ or function of NMDA receptor in the IC caused by tail amputation. Injury-induced
deficits in signaling cascades at the downstream of the NMDA receptor activation may
also contribute to the loss of insular LTD. Regardless of the mechanisms, loss of
the ability to undergo LTD in the IC might be an essential synaptic mechanism accounting
for the maladaptive central plasticity occurring after amputation [25,26,42].

DHPG-induced LTD is not affected by tail amputation

One unexpected finding of this study is that tail amputation did not affect the induction
of DHPG-LTD in superficial and deep layers of the IC. These results stand in contrast
with those obtained from the adult mice ACC slices, where both electrically-induced
LTD and chemically-induced LTD were significantly impaired by tail amputation [38]. The exact reasons for these discrepancies are not clear but might be due to the
differences in the mGluR-targeting drugs used (DHPG vs. DHPG + MPEP) and the forebrain
regions analyzed (IC vs. ACC). The conflicting observations between LFS-and DHPG-induced
insular LTD could arise from their differences in the vulnerability to amputation-elicited
plastic changes in the IC area. This discrepancy is also in accordance with our recent
publication, demonstrating that DHPG-LTD and LFS-induced LTD represent two distinct
forms of LTD co-existing in the insular synapses and do not occlude each other [44].

It is noteworthy that region-related differences might exist when considering the
effects of tissue amputation on synaptic plasticity in the pain-related brain regions
(summarized in Table 1). Specifically, although either tail or digit amputation triggered a complete loss
of LTD in the ACC [37,38] or the IC (the present study), almost the same manipulation has no effect on LTD
induction in the hippocampus or parietal cortex [37,45]. Also, partial ligation of the sciatic nerve, a well-established animal model of
neuropathic pain, does not affect the induction of LFS-evoked LTD in the hippocampus
[46]. These findings indicate that both ACC and IC play important roles in amputation-related
cortical plasticity, and such changes are relatively selective for pain-related areas.
It is unlikely due to the general stress or other non-selective factors caused by
amputation. Targeting these alterations in synaptic plasticity in the brain might
provide an alternative approach for the treatment of chronic pain including the phantom
pain [24,25,42,69].

mGluR-dependent rescue of insular LTD after tail amputation

It is now well-known that mGluRs activation is not only directly involved in the induction
of LTP or LTD, but is also engaged in a process called metaplasticity, by which prior
neuronal activity or mGluRs activation can affect the subsequent ability to exhibit
synaptic plasticity [for reviews, see [61,71-73]. Nevertheless, the current literature mainly indicates the metaplastic role of mGluRs
in facilitation of hippocampal LTP induction, with less emphasis placed upon their
effect on LTD in cortical areas [59,60,74,75]. There are only a few reports showing that priming stimulation of group II mGluRs
inhibits or facilitates the subsequent induction of LTD in CA1 or dentate gyrus, respectively
[76,77], while prior activation of group I mGluRs has no effect [77]. Our recent work in the ACC revealed a facilitatory role of prior mGluR1 activation
on cingulate LTD induction in the tail-amputated mice [38]. Consistently, the present study demonstrated a similar rescue of amputation-impaired
insular LTD by priming treatment with DHPG (20 μM, 20 min). This is the first demonstration
of the metaplasticity phenomenon in the adult mouse IC. More importantly, these observations
highlight the potential of developing mGluR agonists as a novel therapeutic strategy
against phantom pain (see below).

Intracellular protein kinases mediating the pharmacological rescue

In the present study, we also examined the mechanisms of group I mGluR-mediated metaplastic
rescue of insular LTD. Previously, evidence has been obtained to support the role
of PKC in various types of metaplasticity, including NMDA receptor- or prior synaptic
activity-induced subsequent LTP inhibition and LTD facilitation [63,78], mGluRs-mediated LTP enhancement [64,74,79] and inhibition of chemically- or electrically-induced LTD initiation [65,80]. Here, we have added the new findings that PKC activation is also an important contributing
factor governing group I mGluR-mediated metaplastic rescue of amputation-impaired
insular LTD. By contrast, we did not find any participation of PKA or CaMKII in the
process, although there are some previous reports indicating their involvement in
the regulation of metaplasticity [66-68]. Consistently, our previous work found that amputation-induced loss of LTD in the
ACC was rescued by mGluR1-related PKC-dependent mechanisms [38], suggesting the important roles of PKC in mGluR-evoked metaplasticity in both ACC
and IC. It is well known that group I mGluR activation can lead to intracellular calcium
rise and subsequent PKC activation [81,82]. Also, the function of the NMDA receptor can be regulated through PKC-mediated signaling
pathways [74,83,84]. Recently, we reported that the NMDA receptor is involved in the induction of LFS-evoked
LTD in the IC [44]. It is thus reasonable to speculate that bath application of DHPG might result in
significant PKC activation, which then contributes to the restoration of insular LTD
through possible NMDA receptor-related mechanisms in the IC slices from tail-amputated
mice. Importantly, inhibition of PKC did not affect the LTD induction in naïve IC
slices [44], implying that mechanistic differences do exist between synaptic plasticity and metaplasticity
in the IC.

Clinical implications

Phantom pain is a common form of chronic pain syndrome characterized by the feeling
of pain in the missing limb following amputation or deafferentation [25-27]. Until now, the clinical treatment for phantom pain is still limited and inefficient.
Maladaptive plastic changes along the neuroaxis have been proposed to be associated
with the occurrence and intensity of phantom pain [25,31,32,85]. Therefore, reversing these plastic changes may offer a novel way to improve the
treatment of phantom pain or amputation-related brain dysfunctions. Our previous and
present results reveal a loss of LFS-induced LTD in the ACC [38] and IC (the present study) following tail amputation in the adult mice, providing
an alternative mechanism by which peripheral injury elicits long-lasting alterations
in synaptic transmission and function in the central nervous system [37,42,47], also see Table 1]. Furthermore, we demonstrate that priming treatment with DHPG application could
rescue the lost LTD in both ACC and IC after amputation, indicating that drugs acting
at group I mGluRs might hold promise for the rational treatment of phantom pain by
reversing amputation-evoked synaptic dysfunctions in the neocortex. From a clinical
perspective, the multi-synaptic model established in the present study might be useful
for further elucidating synaptic mechanisms of phantom pain in the brain, as well
as screening and developing potential new drugs for treating this intractable disease
in the human amputees.

Methods

Animals

Experiments were performed with adult (7-10 week old) male C57/BL6 mice purchased
from Charles River (Quebec, Canada). All animals were fed in groups of three per cage
under standard laboratory conditions (12 h light/12 h dark, temperature 22-26°C, air
humidity 55-60%) with ad libitum water and mice chow. The experimental procedures were approved by the Institutional
Animal Care and Use Committee of The University of Toronto. All animals were maintained
and cared for in compliance with the guidelines set forth by the International Association
for the Study of Pain [86]. The number of animals used and their suffering were greatly minimized.

Drugs

The drugs used in this study include: DHPG, chelerythrine, KT5720 and KN62. Among
them, chelerythrine and DHPG were dissolved in distilled water, while KT5720 and KN62
were prepared in dimethyl sulfoxide (DMSO) as stock solutions for frozen aliquots
at -20°C. All these drugs were diluted from the stock solutions to the final desired
concentration in the artificial cerebrospinal fluid (ACSF) before immediate use. The
diluted DMSO in ACSF had no effect on baseline synaptic transmission and plasticity.
Chelerythrine, KT5720 and KN62 were purchased from Tocris Cookson (Bristol, UK) and
DHPG was obtained from Abcam Biochemicals (Cambridge, UK). The doses for each compound
were chosen based on our preliminary experiments and on relevant information from
previous papers [38,44,77]. For the pharmacological rescue of insular LTD, DHPG (20 μM) with or without the
drugs was bath applied for 20 min and then washed out for 30 min prior to LTD induction.

Tail amputation

The major procedures for tail amputation are in accordance with those described previously
[38,45,87]. After anesthesia with gaseous isoflurane, the mouse was gently put in a box where
a 2.5 cm length of the tail tip was removed using surgical scissors. A drop of Krazy
Glue was used to stop bleeding. The mice typically recovered from anesthesia within
3-5 min. Amputated animals did not exhibit any neurological deficits or abnormal behaviors
when returned to the home cage. For the sham control group, mice were anesthetized
for the same period of time without any surgery. Procedure was executed with caution
to minimize handling-induced stress in the mice. In the present study, we performed
electrophysiological recordings at 2 weeks after tail amputation (Figure 1A), on the basis of our previous reports showing an evident plastic change in the
ACC at this time point [37-39].

Insular slice preparation

The general procedures for making IC slices are similar to those described previously
[18,23,44,48]. Briefly, mice were anesthetized with a brief exposure to gaseous isoflurane and
decapitated. The entire brain was rapidly removed and immersed into a cold bath of
oxygenated (95% O2 and 5% CO2) ACSF containing (in mM): NaCl 124, KCl 2.5, NaH2PO4 1.0, MgSO4 1, CaCl2 2, NaHCO3 25 and glucose 10, pH 7.35-7.45. After cooling for 1-2 min, appropriate portions
of the brain were then trimmed and the remaining brain block was glued onto the ice-cold
stage of a vibrating tissue slicer (Leika, VT1000S). Following this, three coronal
IC slices (300 μm) were obtained at the level of corpus callosum connection and transferred
to an incubation chamber continuously perfused with oxygenated ACSF at 26°C. Slices
were allowed to recover for at least 2 h before any electrophysiological recording
was started.

Multi-channel field potential recordings

A commercial 64-channel multi-electrode array system (MED64, Panasonic Alpha-Med Sciences,
Japan) was used for extracellular field potential recordings in this study. Procedures
for preparation of the MED64 probe and multi-channel field potential recordings were
similar to those described previously [23,38,43,44]. The device had an array of 64 planar microelectrodes, each 50 × 50 μm in size, arranged
in an 8 × 8 pattern (inter-electrode distance: 150 μm). Before use, the surface of
the MED64 probe was treated with 0.1% polyethyleneimine (Sigma, St. Louis, MO, USA)
in 25 mM borate buffer (pH 8.4) overnight at room temperature. After incubation, one
slice was positioned on the MED64 probe in such a way that the IC area was entirely
covered by the recording dish mounted on the stage of an inverted microscope (CKX41,
Olympus). The relative location of the IC slice with the probe followed the anatomical
atlas [88], also see Figure 1B]. Once the slice was settled, a fine mesh anchor (Warner Instruments, Harvard) was
carefully disposed to ensure slice stabilization during recording. The slice was continuously
perfused with oxygenated, fresh ACSF at the rate of 2-3 ml/min with the aid of a peristaltic
pump (Minipuls 3, Gilson) throughout the entire experimental period.

After a 15-20 min recovery of the slice, one of the 64 available planar microelectrodes
was selected from the 64-switch box for stimulation by visual observation through
a charge-coupled device camera (DP70, Olympus) connected to the inverted microscope.
For test stimulation, monopolar, biphasic constant current pulses (0.2 ms in duration)
generated by the data acquisition software (Mobius, Panasonic Alpha-Med Sciences)
were applied to the deep layer (layer V-VI) of the IC slice at 0.008 Hz (red dot in
Figure 1B). The fEPSPs evoked at both the deep layer and the superficial layer (layer I-III)
of the IC slice were amplified by a 64-channel amplifier, displayed on the monitor
screen and stored on the hard disk of a microcomputer for offline analysis. After
selecting the best stimulation site and stabilizing the baseline synaptic responses,
an input–output curve was first determined for each group using the measurements of
fEPSP slope or the number of activated channels (output) in response to a series of
ascending stimulation intensities from 6 μA to 24 μA by every 2 μA step (input). For
the LTD induction, the intensity of the test stimulus was adjusted to elicit 40-60%
of the maximum according to the input–output curves. Stable baseline responses were
then monitored for at least 20 min before delivering a classical LFS protocol (1 Hz,
900 pulses, with the same intensity as baseline recording) to induce NMDA receptor-dependent
LTD. In another set of experiments, DHPG (100 μM, 20 min) was bath applied to induce
another form of LTD [44]. After LFS or DHPG application, the test stimulus was repeatedly delivered once every
2 min for 1 h or 50 min to monitor the time course of insular LTD.

Data analysis

All multi-channel electrophysiological data were analyzed offline by the MED64 Mobius
software. For quantification of the input–output relationship, the slope of fEPSP
was measured and expressed as the percentage of 8 μA value according to different
layers (superficial layer and deep layer). It is notable that data from the stimulation
intensity of 6 μA were not included in the slope analysis due to the much fewer fEPSP
evoked at this low intensity. The number of activated channels evoked at different
stimulation intensities was also counted in a blind manner. For quantification of
the LTD data, the initial slope of fEPSPs was measured by taking the rising phase
between 10% and 90% of the peak response, normalized and presented separately in both
superficial and deep layers as a percentage change from the baseline level. The degree
of LTD in each experiment was shown as the value obtained at 50 min or 60 min after
DHPG or LFS treatment, respectively. For evaluation of the drug effects on rescued
LTD, the averaged value of the last 10 min of the recording was compared statistically.
Furthermore, the number of activated channels (over 20% of baseline, i.e. the amplitude
goes over -20 μV) vs. the LTD-showing (depressed by at least 15% of baseline) channels
was counted and expressed as the induction ratio of LTD (number of LTD-occurring channels
/number of all activated channels × 100%). All data are shown as mean ± S.E.M. When
necessary, the statistical significance was assessed by two-tailed Student’s t test
and one-way ANOVA (followed by post doc Fisher’s LSD test) using the Sigma Plot software.
P < 0.05 was assumed as statistically significant.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

M.-G.L. performed the experiments, analyzed data and drafted the manuscript; M.Z.
conceived and designed the research and finished the final version of the manuscript.
Both authors read and approved the final manuscript.

Acknowledgements

This work was supported by Canadian Institutes of Health Research (CIHR) operating
grant, Canada Research Chair (CRC), and NSERC (Natural Sciences and Engineering Research
Council of Canada) discovery grant 402555 to MZ.

References

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